Objective To fabricate in situ crosslinking hyaluronic acid hydrogel and evaluate its biocompatibility in vitro. Methods The acrylic acid chloride and polyethylene glycol were added to prepare crosslinking agent polyethylene glycol acrylate (PEGDA), and the molecular structure of PEGDA was analyzed by Flourier transformation infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. Hyaluronic acid hydrogel was chemically modified to prepare hyaluronic acid thiolation (HA-SH). And the degree of HA-SH was analyzed qualitatively and quantitatively by Ellman method. HA-SH solution in concentrations (W/V) of 0.5%, 1.0%, and 1.5% and PEGDA solution in concentrations (W/V) of 2%, 4%, and 6% were prepared with PBS. The two solutions were mixed in different ratios, and in situ crosslinking hyaluronic acid hydrogel was obtained; the crosslinking time was recorded. The cellular toxicity of in situ crosslinking hyaluronic acid hydrogel (1.5% HA-SH and 4% PEGDA mixed) was tested by L929 cells. Meanwhile, the biocompatibility of hydrogel was tested by co-cultured with human bone mesenchymal stem cells (hBMSCs). Results Flourier transformation infrared spectroscopy showed that most hydroxyl groups were replaced by acrylate groups; 1H nuclear magnetic resonance spectroscopy showed 3 characteristic peaks of hydrogen representing acrylate and olefinic bond at 5-7 ppm. The thiolation yield of HA-SH was 65.4%. In situ crosslinking time of hyaluronic acid hydrogel was 2 to 70 minutes in the PEGDA concentrations of 2%-6% and HA-SH concentrations of 0.5%-1.5%. The hyaluronic acid hydrogel appeared to be transparent. The toxicity grade of leaching solution of hydrogel was grade 1. hBMSCs grew well and distributed evenly in hydrogel with a very high viability. Conclusion In situ crosslinking hyaluronic acid hydrogel has low cytotoxicity, good biocompatibility, and controllable crosslinking time, so it could be used as a potential tissue engineered scaffold or repairing material for tissue regeneration.
ObjectiveTo analyze the relationship between the bone mineral density (BMD) and lumbar intervertebral disc degeneration in rhesus macaques by using T1ρ-MRI. MethodsTwenty female rhesus macaques at the age of 10.9 years on average (rang, 4-20 years) were selected. The lumbar intervertebral discs were classified by Pfirrmann grading system and the T1ρ relaxation time (T1ρ value) was examined by using MRI (Philips 1.5 Tesla), and then BMD values of the L4,5 vertebrae and femoral ward's triangle were detected by using Osteocore dual energy X-ray absorptiometry. Finally, the relationship of T1ρ value of the lumbar intervertebral discs and Pfirrmann grading with age, weight, BMD of lumbar vertebrae and femoral ward's triangle was analyzed. ResultsThe BMD values of lumbar vertebrae and femoral ward's triangle were (0.64±0.17) g/cm2 and (0.67±0.19) g/cm2 respectively, showing no significant difference (t=2.893, P=0.128). According to Pfirrmann grading system, there were 7 cases of grade I, 8 cases of grade Ⅱ, and 5 cases of grade Ⅲ at L4,5 intervertebral discs. The T1ρ value of the lumbar intervertebral disc was (104.08±18.65) ms; the T1ρ values of grades I, Ⅱ, and Ⅲ were (121.31±13.44), (104.73±15.01), and (77.41±11.87) ms, respectively. There was a negative correlation between T1ρ value and the age and the BMD of lumbar vertebrae and femoral ward's triangle. There was a positive correlation between Pfirrmann grading and the variables as listed above. Significant negative linear correlation was also observed between T1ρ value and Pfirrmann grading. ConclusionThe T1ρ value is a reliable index when quantifying lumbar intervertebral disc degeneration, and there is a significant positive correlation between BMD and lumbar intervertebral disc degeneration in rhesus macaques.
ObjectiveTo establish the degenerative disc animal model in rhesus macaques and to verify its reliability with T1ρ, spin-lock imaging and T2-mapping MRI and histological observation. MethodsTwelve female rhesus macaques (aged 4-6 years, weighing 4.4-6.1 kg) were enrolled in the study. The L5, 6 intervertebral disk was used for the experimental group by injecting 1 mL bleomycin A5 (2 mg/mL) to its adjacent endplates to induce degeneration, and the L4,5 intervertebral disk for the control group by injecting 1 mL normal sodium to its adjacent endplates. T1ρ and T2-mapping relaxation time was examined by using MRI, and the histological observation was performed to evaluate the process of degeneration at 1, 4, and 12 weeks after operation. ResultsThere was no significant change of T1ρ, T2 map relaxation time in the control group at different time points before and after operation (P>0.05). In the experimental group, there was a significant decrease of T1ρ relaxation time from 4 weeks after operation, showing significant difference when compared with the values at pre-operation and 1 week after operation (P<0.05). The T2 map relaxation time decreased significantly at 12 weeks after operation, showing significant difference when compared with the values at the other time points (P<0.05). There was no significant difference between 2 groups at the other time points (P>0.05) except T1ρ relaxation time at 4 and 12 weeks and T2 map relaxation time at 12 weeks (P<0.05). No significant change of the percentage of the high intensity area of the operated discs was observed on T2WI of MRI after operation in 2 groups (P>0.05). The histological results showed that the number of nucleus pulposus decreased and arranged irregularly at 4 weeks, and there were fibrosis changes of nucleus pulposus and cleft of the inner annulus fibrosus at 12 weeks after operation in the experimental group. ConclusionDegeneration of lumbar intervertebral disc in rhesus macaques can be induced by injecting bleomycin A5. T1ρ-MRI may be an effective method to evaluate early degeneration of intervertebral disc.
ObjectiveTo observe the genes expression of hypoxia inducible factor 1α (HIF-1α) and HIF-2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. MethodsHigh density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2%O2) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type Ⅱ at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type Ⅱ, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. ResultsToluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group;extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type Ⅱ was positive in cytoplasm;when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t=8.345, P=0.001;t=7.683, P=0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P<0.05), but no significant difference was found at 3 weeks between the 2 groups (P>0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P<0.01). The mRNA expressions of collagen type Ⅱ and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P<0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P<0.05). ConclusionHIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.
ObjectiveTo investigate the effect of body mass index (BMI) on the outcome of posterior 360° fusion for single-level lumbar degenerative diseases. MethodsA retrospective study was carried on 302 cases of singlelevel lumbar degenerative diseases treated with posterior 360° fusion between September 2009 and September 2013. All patients were divided into 3 groups according to BMI: normal weight (BMI<24 kg/m2) in 105 cases (group A), overweight (24 kg/m2≤BMI< 28 kg/m2) in 108 cases (group B), and obese (BMI≥28 kg/m2) in 89 cases (group C). There was no significant difference in gender, age, disease duration, disease patterns, affected segments, preoperative Japanese Orthopaedic Association (JOA) score and Oswestry disability index (ODI) among 3 groups (P>0.05). The operation time, intraoperative blood loss, postoperative hospital stay, and complications were recorded. The lumbar function was assessed by JOA score and ODI at pre- and post-operation (at 3, 6, and 24 months). ResultsThe operation time, intraoperative blood loss, and postoperative hospital stay of group C were significantly more than those of groups A and B (P<0.05), but no significant difference was found between group A and group B (P>0.05). The patients were followed up 24-45 months. Postoperative JOA score and ODI showed significant improvements in each group when compared with preoperative ones (P<0.05), but there was no significant difference among groups at each time point after operation (P>0.05). There was no significant difference in the incidence of total complications among 3 groups (χ2=3.288, P=0.193). The incidence of incision-related complications (infection and poor healing) in group C was significantly higher than that of groups A and B (P<0.05), but no significant difference was shown between group A and group B (P>0.05). However, there was no significant difference in cerebrospinal fluid leak, pseudarthrosis formation, and revision among 3 groups (P>0.05). ConclusionPosterior 360° fusion for single-level lumbar degenerative diseases can obtain good effectiveness in patients with different BMI, but patients whose BMI was ≥28 kg/m2 have longer operation time, more intraoperative blood loss, longer hospital stay, and higher incidence of postoperative incision-related complications.
ObjectiveTo explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice. MethodsMC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I). ResultsThe results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P < 0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P < 0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P < 0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). ConclusionThe appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.